Α-oxo Acids Research Articles

HPLC−MS/MS Profiling of Tryptophan-Derived Alkaloids in Food: Identification of Tetrahydro-β-carbolinedicarboxylic Acids

A method for selective detection of 1,2,3,4-tetrahydro-β-carbolinecarboxylic acids (THCCs) was developed based on electrospray ionization−tandem mass spectrometry coupled to liquid chromatography (HPLC−ESI−MS/MS). Low-energy collision-induced dissociation (CID) led to characteristic fragment ions due to neutral loss of 73 amu. Subsequently, constant neutral loss scanning was used for substructure specific screening of THCCs in food samples. Detection limits for HPLC−ESI−MS/MS analysis of THCCs applying neutral loss experiments were established at 100 ng mL-1 (ca. 2.5 pmol on column). Application of this MS/MS method enabled us to detect THCC derivatives derived from Pictet−Spengler condensation of tryptophan with α-oxo acids. Subsequently, diastereomeric 1,2,3,4-tetrahydro-β-carboline-1,3-dicarboxylic acid 3a/b, 1-methyl-1,2,3,4-tetrahydro-β-carboline-1,3-dicarboxylic acid 4a/b, and 1-(2‘-carboxyethyl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid 5a/b were identified in alcoholic beverages, seasoning ...

Primordial Amino Acids by Reductive Amination of α-Oxo Acids in Conjunction with the Oxidative Formation of Pyrite

Primordial Amino Acids by Reductive Amination of α-Oxo Acids in Conjunction with the Oxidative Formation of Pyrite

Primordial amino acids by reductive amination of α-oxo acids in conjunction with the oxidative formation of pyrite

Abstract The theory of an autotrophic origin of life postulates a primordial formation of amino acids by a mild and specific chemical energy source, namely by the reductive amination of α-oxo acids in conjunction with the oxidative formation of pyrite. Here we show experimental proof for this reaction, which involves carbon dioxide as catalyst.

ChemInform Abstract: Photochemical Fixation of Carbon Dioxide: Enzymic Photosynthesis of Malic, Aspartic, Isocitric, and Formic Acids in Artificial Media.

Photosensitized regeneration of 1,4-dihydronicotinamide adenine dinucleotide phosphate (NADPH) with an artificial photosystem allows the enzymic fixation of CO2 through carboxylation of α-oxo acids using sacrificial electron donors. Pyruvic acid is carboxylated to malic acid and α-oxoglutaric acid is carboxylated to isocitric acid with the malic enzyme and isocitrate dehydrogenase (ICDH) as biocatalysts, φ= 1.9%. Malic acid formed through the photosensitized process is used as a synthetic building block for subsequent sesquestered enzymic transformation, and its conversion into aspartic acid is accomplished with fumarase and aspartase as biocatalysts.Photoreduction of CO2 to formate is accomplished in the presence of formate dehydrogenase (FDH) as catalyst. Photosensitized reduction of different bipyridinium relay systems, i.e. N,N′-dimethyl-4,4′-bipyridinium (MV2+)(1), N,N′-dimethyl-2,2′-bipyridinium (DM2+)(2), N,N′-trimethylene-2,2′- bipyridinium (DT2+)(3), and N,N′-tetramethylene-2,2′-bipyridinium (DQ2+)(4), to the corresponding radical cations yields reduced relays that act as cofactors for FDH, which mediates the reduction of CO2 to formate. The quantum yield for formate formation is in the range φ= 0.5–1.6%

Photochemical fixation of carbon dioxide: enzymic photosynthesis of malic, aspartic, isocitric, and formic acids in artificial media

Photosensitized regeneration of 1,4-dihydronicotinamide adenine dinucleotide phosphate (NADPH) with an artificial photosystem allows the enzymic fixation of CO2 through carboxylation of α-oxo acids using sacrificial electron donors. Pyruvic acid is carboxylated to malic acid and α-oxoglutaric acid is carboxylated to isocitric acid with the malic enzyme and isocitrate dehydrogenase (ICDH) as biocatalysts, φ= 1.9%. Malic acid formed through the photosensitized process is used as a synthetic building block for subsequent sesquestered enzymic transformation, and its conversion into aspartic acid is accomplished with fumarase and aspartase as biocatalysts.Photoreduction of CO2 to formate is accomplished in the presence of formate dehydrogenase (FDH) as catalyst. Photosensitized reduction of different bipyridinium relay systems, i.e. N,N′-dimethyl-4,4′-bipyridinium (MV2+)(1), N,N′-dimethyl-2,2′-bipyridinium (DM2+)(2), N,N′-trimethylene-2,2′- bipyridinium (DT2+)(3), and N,N′-tetramethylene-2,2′-bipyridinium (DQ2+)(4), to the corresponding radical cations yields reduced relays that act as cofactors for FDH, which mediates the reduction of CO2 to formate. The quantum yield for formate formation is in the range φ= 0.5–1.6%

XY–ZH systems as potential 1,3-dipoles. Part 6. Metallo-1,3-dipoles. Cycloadditions of divalent metal complexes of glycine and alanine imines to electronegative olefins

Copper(II), zinc(II), and cadmium(II) complexes of imines derived from α-oxo acids and glycine or alanine undergo stereospecific cycloaddition at room temperature to 1,2-disubstituted electronegative olefins in the presence of weak base. Corresponding reactions of lead(II) complexes give insoluble cycloadducts. In contrast, the reactions of the same metallo imines with methyl acrylate, phenyl vinyl sulphone, and acrylonitrile frequently give mixtures of regio- and stereo-isomers. These mixtures are shown to arise by stereoisomerisation of initial cycloadducts formed by a 4π+ 2π concerted cycloaddition of intermediate metallo-1,3-dipoles.

Characterization of artefacts produced by treatment of organic acids with diazomethane

When α,β-unsaturated acids and α-keto acids react with diazomethane not only are the corresponding methylates produced, but also diazomethane is added to the CC double bond or to the oxo group. The gas chromatographic and mass spectral behaviour of these undesired products and some further artefacts produced in the hot inlet lines of a gas chromatograph are described. The mass spectra and retention indices allowed the structural assignment of several “unknown” compounds found previously in the methylated acid fraction of urine. A detailed analysis of the reaction of α-oxo acids with diazomethane revealed that, besides the already known oxirane methyl esters, homologous esters are also produced by an insertion reaction.

A novel way for the formation of α-amino acids and their derivatives in an aqueous medium

In the course of a study of possible mechanism for chemical evolution in the primeval sea, we observed the formation of α-amino acids and N-acylamino acids from α-oxo acids and ammonia in an aqueous medium. Glyoxylic acid reacted with ammonia to form N-oxalylglycine, which gave glycine in a 5–39% yield after hydrolysis with 6N HCl. Similarly when glyoxylic acid was treated with methylamine it yielded N-oxalylsarcosine, which could be hydrolyzed to sarcosine with 17–25% overall yield upon hydrolysis. Pyruvic acid and ammonia reacted to give N-acetylalanine, which formed alanine in a 3–7% overall yield upon hydrolysis. The pH optima in these reactions were pH 3–4. These reactions were further extended to the formation of other amino acids. Glutamic acid, phenylalanine and serine were formed from α-ketoglutaric acid, phenylpyruvic acid and hydroxypyruvic acid, respectively, under similar conditions, N-Succinylglutamic acid was obtained as an intermediate for glutamic acid synthesis. Phenylacetylphenylalanineamide was also isolated as an intermediate for phenylalanine synthesis. Alanine, rather than aspartic acid, was produced from oxaloacetic acid. These reactions provide a novel route for the prebiotic synthesis of amino acids. A mechanism for the reactions is proposed.

Effects of selected analogs of the branched-chain α-oxo acids on the α-oxo acid dehydrogenase activities in rat liver mitochondria

Various cyclic alkaneglyoxylic acids and 1-hydroxy-cyclicalkanecar-boxylic acid analogs of the branched-chain oxo acids were synthesized and tested along with some commercially available analogs as inhibitors for the branched-chain α-oxo acid dehydrogenase enzyme system as well as for the pyruvate and α-oxoglutarate dehydrogenase enzyme systems. Cyclopentaneglyoxylic acid and cyclohexaneglyoxylic acid gave the most promising results in terms of specifically inhibiting the branched-chain α-oxo acid dehydrogenases.

水溶液中でのα‐オキソ酸からのα‐アミノ酸の形成

In the course of experimental studies on chemical evolution in primeval sea, we discovered a novel way for amino acid formation via N-acylamino acid from an aqueous mixture of α-oxo acid and ammonium sulfate. Glycine and alanine were produced from glyoxylate and pyruvate through N-oxalylglycine and N-acetylalanine, respectively. The process was extended to the formation of other amino acids. Serine, glutamic acid, and phenylalanine were produced from hydroxypyruvate, α-ketoglutarate, and phenylpyruvate, respectively, in aqueous medium under similar conditions. In the case of glutamic acid formation, N-succinylglutamic acid, which was partially converted to pyroglutamic acid was found as an intermediate. However, in the case of phenylalanine formation, N-phenylacetylphenylalanineamide (mp 190_??_191°C) was isolated and identified by IR (3170, 1660, 1640, 1530cm-1), NMR [δ (ppm)=7.18 (10 H, s), 4.45 (1 H, m), 3.38 (2 H, s), 3.20_??_2.60 (2 H, m)], MS [282 (M+), 238], and traditional synthesis, as an intermediate, instead of N-phenylacetylphenylalanine. From oxaloacetate in similar conditions, alanine was produced instead of the expected aspartic acid. In these reactions, the decarboxylation processes were essential for yielding N-acylamino acids when employing α-oxo acids except for glyoxylic acid (R-CO-COOH, R_??_H).

Fluorescence and chemiluminescence assay of biological substances.

Highly sensitive analytical methods of various biological substances developed in the author's laboratory are reviewed. Amino sugarsand amino acids are determined by a fluorophotometric method using pyridoxal-Zn(II) ion. α-Oxo acids are also assayed fluorophotometrically with pyridoxamine-Zn(II) ion. Various oxosteroids are assayed by fluorescence high-performance liquid chromatography using dansyl hydrazine as a pre-labeling reagent. Bile acids are also determined fluorophotometrically with highperformance liquid chromatography after labeling with 1-bromoacetylpyrene. Fluorescence and chemiluminescence reactions are applied to enzyme immunoassays of various hormones and drugs.

Biochemical gastroprotection from acute ulceration induced by aspirin and related drugs

Adjuncts that serve as: (a) buffers to neutralize drug acidity, and/or (b) solubilizers of acidic drugs, or (c) certain nutrients (e.g. glucose, acetate), considerably reduced the gastric mucosal injury induced by orally administered aspirin (and other non-steroidal anti-inflammatory drugs) in stressed and starved rats. Gastroprotection against aspirin and related drugs was obtained by supplying the mucosa with glucose with intermediates or precursors of the tricarboxylic acid cycle (that may be absorbed directly from the gastric lumen). Glucose alone was not sufficiently gastroprotective. Gastroprotection with tricarboxylic acid cycle precursors given with glucose appears to be due to the effects of these nutrients in restoring ATP synthesis in the gastric mucosa. d-glutamate and d-aspartate were deaminated by homogenates prepared from saline-washed rat fundic mucosa (yielding α-oxo acids for the tricarboxylic acid cycle). These amino acids could be substituted for the l-forms in combination with glucose to yield gastroprotection from damage by aspirin. Studies in domestic pigs (a species with a pseudo-human stomach) established that acute and chronic oral administration of the aspirin + acetate + glucose combination (1: 3: 3 molar proportions) was less irritating to the gastric mucosa than aspirin alone. These results were confirmed in acute studies in monkeys. Sodium and potassium salts were superior to calcium and ammonium salts as the buffer component in these improved (i.e. less gastrotoxic) aspirin formulations tested in rats. Bicarbonate was not effective in preventing aspirin gastrotoxicity in stressed-sensitized rats, but is effective in non-stressed rats.

A fluorophotometric method for the determination of α-oxo acids by using pyridoxamine and zinc(II) ion

A fluorophotometric method for the determination of α-oxo acids has been developed based on their interaction with pyridoxamine and Zn(II) ion in pyridine-methanol solution to yield highly fluorescent chelates. This fluorophotometric procedure is simple and highly sensitive to α-oxo acids. No detectable interference for the determination of pyruvate occurred with any of the amino acids, amines, sugars, organic acids, and inorganic salts. Recoveries for pyruvate added to bloods and serum ranged from 90.1 to 103.2%. As little as 5 × 10 −11 mol of α-oxo acid may be measured.

The purification and properties of glutamine-dependent asparagine synthetase isolated from Lupinus albus

Glutamine-dependent asparagine synthetase, which transfers the amide nitrogen of glutamine to aspartate has been purified from Lupinus albus . The enzyme has a very high affinity for glutamine although it is able to use ammonia as a substrate. The enzyme is inhibited by α-oxo acids, in particular α-oxoglutarate; the possible physiological role is discussed. The activities of various other enzymes involved in ammonia assimilation have been measured during development of the cotyledon. A pathway of the route of ammonia entry into various amino acids has been proposed. The substrate specificity of the enzyme has been investigated with analogues of glutamine, aspartate and asparagine.

Purification and properties of dolphin muscle aspartate and alanine transaminases and thier possible roles in the energy metabolism of diving mammals.

1. Mitochondrial and supernatant aspartate transaminases (EC 2.6.1.1) and supernatant alanine transaminase (EC 2.6.1.2) were purified 89-, 204- and 240-fold respectively, from dolphin muscle. Starch-gel electrophoresis of crude and purified preparations revealed that all three enzymes exist as single forms. 2. K(m) values of alpha-oxoglutarate, alanine, pyruvate and glutamate for the alanine transaminase were 0.45, 8.2, 0.87 and 15mm respectively. For the aspartate transaminases, the K(m) values of alpha-oxoglutarate, aspartate, oxalacetate and glutamate were 0.76, 0.50, 0.10 and 9.4mm respectively, for the mitochondrial form and 0.13, 2.4, 0.06 and 3.2mm respectively, for the supernatant form. 3. In all cases, as the assay pH value was decreased from pH7.3, the K(m) values of the alpha-oxo acids decreased whereas those of the amino acids increased. 4. The apparent equilibrium constants for the aspartate transaminases were independent of pH. These values were 9.2 and 6.8 for the mitochondrial and supernatant forms respectively, where [Formula: see text] 5. Studies of the inhibition of the aspartate transaminases by dicarboxylic acids indicated that these enzymes may be controlled by pools of metabolic intermediates. 6. Three key roles are suggested for the transaminases in the energy metabolism of the diving animal. First, it is believed that a combined action of the transaminases could enhance energy production during hypoxia by providing (a) fumarate from aspartate for the ATP-producing reversal of succinate dehydrogenase, and (b) alpha-oxoglutarate from glutamate for the GTP-producing succinyl thiokinase reaction. Secondly, diving mammals probably accumulate more NADH than other mammals during hypoxia. The aspartate transaminases seem particularly well suited for restoring and maintaining redox balance via the malate-aspartate cycle after aerobic metabolism is resumed. Finally, since the preferred fuel for aerobic work is fat, the combined reactions of the transaminases could be instrumental in providing increased supplies of oxaloacetate for sparking the tricarboxylic acid cycle.

Endogenous alanine, glutamate, aspartate, and glutamine in the perfused guinea-pig heart: effect of substrates and cardioactive agents.

The isolated, nonworking guinea-pig heart was perfused with 14C-labelled glucose, pyruvate, and acetate. Labelled and total alanine, glutamate, aspartate, and glutamine were measured. The alanine content of the heart varied directly with the medium pyruvate concentration. The sum of the concentrations of glutamate and aspartate varied inversely with the alanine concentration. The sum of alanine, glutamate, aspartate, and glutamine in the heart was constant under most perfusion conditions, but loss into the medium increased in the presence of ouabain, low Ca2+ concentration, or malonate. The sum of the total amino acids from the heart and medium remained constant under all conditions except in the presence of malonate, when it was decreased.Labelling of amino acids from 14C-labelled substrates indicated that alanine, glutamate, and aspartate exchanged readily with their α-oxo acids. However, only 30% of the glutamine exchanged with glutamate during 1 h; this proportion varied little with perfusion conditions or the metabolic flux. The results indicate that in a closed system, most of the changes in the concentrations of amino acids are brought about by transamination.

The influence of steric and electronic parameters on the substrate behavior of -oxo acids to yeast pyruvate decarboxylase.

The Michaelis constants of yeast pyruvate decarboxylase at full substrate activation and the maximum decarboxylation rates were determined from a total of 21 aliphatic and aromatic α‐oxo acids in the absence of buffer substances. By means of a pyruvamide‐activated enzyme it was possible to confirm experimentally the Km‐values obtained by extrapolation. The substrate binding rate of pyruvate decarboxylase increases linearly with increasing H+ concentration, which indicates that only the undissociated pyruvic acid acts as the substrate of the enzyme. Whilst the substrate binding rate can be correlated linearly with the steric substituent constants of aliphatic α‐oxo acids, it was not possible to find a corresponding relation between the aromatic α‐oxo acids and the Hammett σ‐constants, which indicates steric peculiarities in the active centre of pyruvate decarboxylase. On the other hand, a linear relation between the maximum velocities and the σ+‐values for direct conjugation was established, leading to some conclusions concerning the mechanism of the splitting‐off of aldehyde.

Zur theorie der α-ketosäuren : Kryoskopische untersuchungen der assoziation aliphatischer α-ketosäuren und phenylglyoxylsäuren in benzol

The type of association of aliphatic α-oxo acids, RCOCOOH, in benzene depends on the alkyl substituent R. Since this effect has also been observed in p-alkyl-phenylglyoxylic acids, it cannot be explained in terms of intramolecular steric interactions but is due to the variation in the lyophilic behaviour of the dissolved molecules.

Kinetic properties of the partially purified pyruvate dehydrogenase complex of ox brain.

The properties of a purified preparation of the pyruvate dehydrogenase complex from ox brain have been compared with those of a similar preparation from ox kidney. A broad pH optimum around 7.8, similar dependence on ionic strength, and independence of the nature of the buffer anions or cations characterized preparations from both tissues. Michaelis constants for the binding of pyruvate, thiamin pyrophosphate, NAD(+) and CoA were also similar. Enzyme from both tissues was inhibited by NADH, by copper and other heavy metals, by high concentrations of tricarboxylic acid-cycle intermediates, and by preincubation with ATP. Acetyl-CoA itself did not appear to inhibit these preparations, although some commercial preparations of acetyl-CoA did contain an inhibitor. Although oxaloacetate and alpha-oxobutyrate were weak inhibitors, a number of other alpha-oxo acids including phenylpyruvate did not inhibit. The properties of the pyruvate dehydrogenase complex from brain and kidney appeared similar.

The oxidation of Schiff bases of pyridoxamine with -oxo acids by manganous ions and peroxidase.

1. Oxygen was taken up rapidly when pyruvate was added to mixtures of pyridoxamine and Mn(2+) ions after lag periods that were shortened by peroxidase (donor-hydrogen peroxide oxidoreductase, EC 1.11.1.7). 2. The total oxygen uptake was proportional to the pyridoxamine added and was accompanied by the disappearance of pyridoxamine; the pyruvate acted catalytically and hydrogen peroxide was not formed. 3. At pH6 more than half the pyridoxamine that disappeared was accounted for as pyridoxal and ammonia; it is suggested that the primary reaction is the oxidative deamination of the pyridoxamine. 4. Results were similar when alpha-oxobutyrate or glyoxylate were substituted for pyruvate, except that the reactions were slower and the yield of pyridoxal less. 5. The oxidative decarboxylations of alpha-oxoglutarate and phenylpyruvate are catalysed by Mn(2+) ions and these reactions are activated by peroxidase; pyridoxamine increased both the rates and total oxygen uptakes in these reactions, and ammonia was produced. 6. The lag periods in the oxidation of mixtures of pyridoxamine and alpha-oxo acids, catalysed by Mn(2+) ions, were also shortened by traces of colloidal manganese dioxide. 7. It is suggested that the activating effect of peroxidase depends on its catalysis of manganese oxidation.